Roll over eggs…it’s time for (unrolled) tobacco leaves

Tobacco leaf infected with Tobacco Mosaic Virus. Courtesy of Clemson University - USDA Cooperative Extension Slide Series

Timeline, 2008: If you’ve ever been asked about allergy to egg products before receiving a flu vaccine, you have had a little encounter with the facts of vaccine making. Flu viruses to produce the vaccine are painstakingly grown in chicken eggs because eggs make perfect little incubators for the bugs.

So…many…eggs

There are problems—in addition to the allergy issue—that arise with this approach. First of all, growing viruses for a million vaccine doses usually means using a million fertilized, 11-day-old eggs. For the entire population of the United States, 300 million eggs would be required. Second, the process requires months of preparation, meaning a slow turnaround time for vaccines against a fast-moving, fast-changing disease. Last, if there is anything wrong with the eggs themselves, such as contamination, the whole process is a waste and crucial vaccines are lost.

The day may come when we can forget about eggs and turn to leaves. Plants can contract viral disease just like animals do. In fact, an oft-used virus in some research fields is the tobacco mosaic virus, which, as its name implies, infects tobacco plants. It gives a patchy look to the leaves of infected plants, and researchers use this feature to determine whether the virus has taken hold.

Bitter little avatars of evil used for good?

Tobacco plants themselves, bitter little avatars of evil for their role in the health-related effects of smoking, serve a useful purpose in genetic research and have now enhanced their approval ratings for their potential in vaccine production. Plants have caught the eye of vaccine researchers for quite a while because they’re cheaper and easier to work with than animal incubators. Using plants for quick-turnaround vaccine production has been a goal, but a few problems have hindered progress.

To use a plant to make a protein to make a vaccine, researchers must first get the gene for the protein into the plant. Previous techniques involved tedious and time-consuming processes for inserting the gene into the plant genome. Then, clock ticking, there was the wait for the plant to grow and make the protein. Add in the Byzantine process of obtaining federal approval to use a genetically modified plant, and you’ve got the opposite of “rapid” on your hands.

One solution to this problem would simply be to get the gene into the plant cell cytoplasm for immediate use. It’s possible but involves meticulously injecting a solution with the gene sequence into each leaf. Once the gene solution is in, the plant will transcribe it—copy it into mRNA—in the cell cytoplasm and then build the desired protein based on the mRNA code. But there has been no way to take hand injection to the large-scale production of proteins, including for vaccines.

Age-old vacuum suction =  high-tech high-throughput

To solve this problem, researchers turned to one of our oldest technologies: vacuum suction. They grew tobacco plants to maturity and then clipped off the leaves, which they submerged in a solution. The solution was spiked with a nasty bug, Agrobacterium tumefaciens, a pathogen responsible for the growth of galls, or tumors, on plants. Anyone working in agriculture fears this bacterium, a known destroyer of grapes, pitted fruit trees, and nut trees. But it does have one useful feature for this kind of work: It can insert bits of its DNA into plant cells. The researchers tricked A. tumefaciens into inserting another bit of DNA instead, the code for the protein they wanted to make.

To get the solution close to the cells, the investigators had to get past air bubbles, and that’s where the vacuum came in. They placed the submerged leaves into a vacuum chamber and flipped a switch, and the activated chamber sucked all the air out of the leaves. When the vacuum was turned off, the solution flowed into the now-empty chambers of the leaf, allowing the A. tumefaciens-spiked solution to bathe the plant cells. After 4 days and a few basic protein-extraction steps, the research team had its protein batch. According to the team lead, “any protein” could be made using this process, opening up almost unlimited possibilities and applications for this approach.

Vaccines…or combating bioterrorism?

The technology has come far enough that a US company has taken steps toward manufacturing vaccines using tobacco leaves.  And it appears that the applications go beyond vaccines, as one news story has noted…the tobacco plants might also be used to produce antidotes to common agents of bioterrorism.

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Microbes redirect our best-laid plans

Greeks at War (pottery from the British Museum; photo courtesy of Wikimedia Commons)

The madness of King George

Timeline, 2006: It’s not that unusual for disease to alter the course of history–or, history as humans intended it to be. Some scholars believe, for example, that the intransigence of King George III of England arose from his affliction with porphyria, a heritable metabolic disorder that can manifest as a mental problem, with symptoms that include irrational intractability. But it’s rarer for a disease to shift the balance of power so entirely that one nation gains the upper hand over another. Yet that appears to be what happened to the city-state of Athens just before the Peloponnesian Wars of around 430 B.C. The upshot of the wave of disease and the wars was that Sparta conquered Athens in 404 B.C.

Spartans had a little microbial assistance

Sparta may have owed its big win to a small bacterium, Salmonella enterica enterica serovar Typhi, the microbe responsible for typhoid fever. A plague swept across Athens from 430 to 426 B.C., having traveled from Ethiopia to Egypt and Libya before alighting in the Greek city and destroying up to a third of its population. In addition, it brought to a close what became known as the Golden Age of Pericles, a time when Athens produced some of its most amazing and widely recognized art, artists, and philosophers, including Aeschylus, Socrates, the Parthenon, and Sophocles. Pericles was a statesman who oversaw the rebuilding of Athens following the Greek win in the Persian Wars, and he guided the city-state to a more democratic form of rule and away from the dictatorships of the previous regimes. In the process, the city flourished in art and architecture.

And then along came the plague. The Greek historian Thucydides, who chronicled the Peloponnesian Wars, left behind such a detailed account of the plague, its symptoms, and what happened to its victims, that intrigued medical detectives have ever since debated about what might have caused it. Thucydides, himself a plague survivor, vividly described the sudden fever, redness of the eyes, hemorrhaging, painful chest and cough, stomach distress, and diarrhea that ultimately led to death in so many cases. He also mentioned pustules and ulcers of the skin and the loss of toes and fingers in survivors. This litany of symptoms produced many candidate causes, including bubonic plague, anthrax, smallpox, measles, and typhoid fever.

Construction crews yet again uncover something interesting

In the mid-1980s, a construction crew was busy digging a hole for a subway station in the city of Kerameikos when they uncovered a mass burial site. Unlike other Greek burial sites, this one bore marks of hasty and haphazard burials, and the few artefacts that accompanied the bones dated it to the time of the plague that destroyed Athens. Researchers were able to harvest some teeth from the site and analyze the tooth pulp, which retains a history of the infections a person has suffered. They examined DNA sequences from the pulp for matches with suggested microbial agents of the plague, and finally found a match with the typhoid bacterium.

Typhoid Mary: Intent on cooking, ended up killing

One discrepancy between the disease pattern of typhoid fever and that described by Thucydides is the rapidity of onset the Greek historian detailed. Today, typhoid fever, which still infects millions of people worldwide, takes longer to develop in an infected individual, and sometimes never develops at all. People who bear the virus but don’t become ill themselves are “carriers.”

Perhaps the most famous carrier was Typhoid Mary, Mary Mallon, a cook in New York City at the beginning of the 20th century. It is believed that she infected hundreds of people, with about 50 known cases and a handful of deaths being directly associated with her. Typhoid Mary was told not to work as a cook any longer or she would be quarantined, but she simply disappeared for awhile and then turned up under a different name, still working as a cook. After another outbreak was traced to her, she was kept in quarantine for 23 years until she died.

Your mother *is* always with you

Mother and child, microchimeras

When you’re in utero, you’re protected from the outside world, connected to it only via the placenta, which is supposed to keep you and your mother separated. Separation is generally a good thing because you are foreign to your mother, and she is foreign to you. In spite of the generally good defenses, however, a little bit of you and a little bit of her cross the barrier. Scientists have recently found that when that happens, you often end up toting a bit of mom around for decades, maybe for life.

The presence of cells from someone else in another individual is called microchimerism. A chimera in mythology was a beast consisting of the parts of many animals, including lion, goat, and snake. In genetics, a chimera carries the genes of some other individual along with its own, perhaps even the genes of another species. In microchimerism, we carry a few cells from someone else around with us. Most women who have been pregnant have not only their own cells but some cells from their offspring, as well. I’m probably carrying around cells from each of my children.

Risks and benefits of sharing

Microchimerism can be useful but also carries risks. Researchers have identified maternal cells in the hearts of infants who died from infantile lupus and determined that the babies had died from heart block, partially from these maternal cells that had differentiated into excess heart muscle. On the other hand, in children with type 1 diabetes, maternal cells found in the pancreatic islets appear to be responding to damage and working to fix it.

The same good/bad outcomes exist for mothers who carry cells from their children. There has long been an association between past pregnancy and a reduced risk of breast cancer, but why has been unclear. Researchers studying microchimerism in women who had been pregnant found that those without breast cancer had fetal microchimerism at a rate three times that of women who with the cancer.

Microchimerism and autoimmunity

Autoimmune diseases develop when the body attacks itself, and several researchers have turned to microchimerism as one mechanism for this process. One fact that led them to investigate fetal microchimerism is the heavily female bias in autoimmune illness, suggesting a female-based event, like pregnancy. On the one hand, pregnancy appears to reduce the effects of rheumatoid arthritis, an autoimmune disorder affecting the joints and connective tissues. On the other hand, women who have been pregnant are more likely to develop an autoimmune disorder of the skin and organs called scleroderma (“hard skin”) that involves excess collagen deposition. There is also a suspected association between microchimerism and pre-eclampsia, a condition in pregnancy that can lead to dangerously high blood pressure and other complications that threaten the lives of mother and baby.

Human leukocyte antigen (HLA)

The autoimmune response may be based on a similarity between mother and child of HLA, immune-related proteins encoded on chromosome 6. This similarity may play a role in the immune imbalances that lead to autoimmune diseases; possibly because the HLAs of the mother and child are so similar, the body clicks out of balance with a possible HLA excess. If they were more different, the mother’s immune system might simply attack and destroy fetal HLAs, but with the strong similarity, fetal HLAs may be like an unexpected guest that behaves like one of the family.

Understanding the links between microchimerism and disease is the initial step in exploiting that knowledge for therapies or preventative approaches. Researchers have already used this information to predict the development of a complication in stem cell transplant called “graft-versus-host disease” (GVH). In stem cell transplants, female donors with previous pregnancies are more associated with development of GVH because they are microchimeric. Researchers have exploited this fact to try to predict whether or not there will be an early rejection of a transplant in kidney and pancreas organ transplants.

(Photo courtesy of Wikimedia Commons and photographer Ferdinand Reus).

Beethoven died of lead poisoning–or did he?

Did lead kill Beethoven?

Timeline, 2005 and 2010: Literary folk have often noted the passion and emotion of Ludwig van Beethoven’s works. Lucy Honeychurch, the heroine of E.M. Forster’s A Room with a View, became “peevish” after playing Beethoven, and of course there’s the famous hooligan Alex from A Clockwork Orange, who was roused to stunning displays of violence after hearing “Ludwig van.” Given Beethoven’s own behavior, which was punctuated by violent rages, frequent sudden outbursts, and wandering the streets humming loudly, it’s not surprising that his music would communicate his passion.

A heavy metal influence?

A study in 2005 (news release here) yielded results that suggested that much of his anger, however, was attributable to the effects of heavy metal…specifically, lead. Beethoven became sick in his 20s (he also went deaf in his 20s), and suffered until his death at the age of 56 from a variety of illnesses, including chronic diarrhea and other stomach ailments. His death was lingering and painful, and some people thought that he had suffered from syphilis. Yet now many of his symptoms fit the classic description of slow lead poisoning. Among the effects of lead poisoning are irritability, aggressive behavior, headaches, and abdominal pain and cramping, all of which Beethoven experienced.

Doctor to businessmen to Sotheby’s to science

Some samples of the great composer’s hair and skull are available today for sophisticated testing for metals. A Viennese doctor apparently snagged a few fragments of his skull 142 years ago and the pieces eventually made their way through the family to a California businessman. The hairs were cut by a student soon after Beethoven died and ended up at a Sotheby’s auction. A few years ago, tests on the hairs suggested that Beethoven’s body harbored high levels of lead—hair accumulates and retains such toxins better than any other tissue—but because the testing method destroyed the hair, further tests were not completed.

Wobbling electrons solve the mystery?

Since that time, a powerful new X-ray technique has become available. The Department of Energy’s Argonne National Laboratory owns the X-ray. In the facility, subatomic particles fly through a tubular tunnel almost at the speed of light, emitting as they travel X-rays 100 times brighter than the sun’s surface. These X-rays can bounce off of the surface of even a tiny sample. As they bounce off of the sample, electrons wobble out of place, releasing energy in a pattern that is specific to the atom being bombarded.

Researchers were interested in Beethoven’s hair and skull pieces. The team that evaluated the samples actually works on developing bacteria that can take up heavy metals and render them relatively harmless; such organisms would be useful in environmental detoxification. They placed Beethoven’s hair in their high-powered X-ray. The electrons wobbled and the pattern indicated that Beethoven was simply full of lead. In fact, they reported that the poor man had about 60 parts per million of lead in his body, which is 100 times normal levels. It certainly was enough to make a person manifest the various symptoms that characterized most of Beethoven’s life.

The team also looked for a pattern that arsenic would emit, and they found none. This result seemed to exonerate Beethoven from having had syphilis, since arsenic would have been the treatment of choice for such an ailment.

Not so fast

At the time the study results were revealed, ideas about how did Beethoven built up so much lead abounded. Some suggested that  his body was less able than normal to rid itself of the heavy metal, through which he’d have been exposed by many channels. His stomach problems and temperament led him to consume much wine, and the vessels for drinking wine contained lead. In addition, his medicines probably were stored in lead-lined bottles or vials, and he may well have visited spas—for his health, ironically—at which he consumed or swam in mineral water containing lead. In one report, Beethoven’s poor doctor was identified as the likely culprit in his demise.

Fast-forward five years to 2010. A deeper analysis (news release here) of the bone fragments from Beethoven’s school indicated that his lead levels were not that spectacular. The bone is the reservoir for most of the lead the body takes up, and Beethoven’s bones simply didn’t have enough to have caused his various physical ailments. While the experts seemed to be in agreement that the results point away from lead, a new heavy metal mystery arose from the results. One skull fragment they tested had about 13 mcg of lead per gram of bone, nothing to write home about, while another sample turned up with 48 micrograms per gram, a much higher level. Nevertheless, we must look elsewhere for what killed one of the world’s greatest western composers. Ideas being tossed around include lupus and heart disease. What we do know is that he lived in terrible pain, both from his maladies and from the treatments designed to help, including pouring hot oil in his ears, according to one Beethoven scholar quoted in the New York Times.

Another wrongly accused suspect

Heavy metals have featured in other historical whodunits. For example, Napoleon reportedly died of stomach cancer during his exile on Elba, but one analysis showed that he actually died of slow arsenic poisoning, suggested to have been at the hand of his closest assistant. Then, much like Beethoven’s story, a later study showed that arsenic likely played no role in the great general’s death.

Crazy cat lady may have microbe to blame

Toxoplasma gondii is the cat-borne parasite responsible for causing toxoplasmosis and a host of other problems in humans. This close relative of the malaria-causing protozoan may drive human behavior and immunity, in addition to causing acute illness and devastating birth defects. Recent research points to a single gene underlying this parasite’s virulence in the human host. It’s scary yet fascinating to think that a single gene from a single organism could have such dramatic effects on our species.

Warning pregnant women away from litter boxes

Because T. gondii infection can result in serious fetal defects, many pregnant women have heard of toxoplasmosis, an illness that often goes unnoticed in the afflicted person. Pregnant women are warned away from cat litter boxes and even away from gardening because contact with cat feces can mean contact with the parasite. T. gondii spends the sexual part of its life cycle in cats, but for its asexual life, it can parasitize a number of hosts, from pigs to lambs to mice to people. People also can acquire the infection from eating undercooked meat or drinking contaminated water. In some countries, like Brazil, up to 60% of the population has been exposed to T. gondii; in the United States, about 33% of people tested have antibodies to the parasite, indicating past infection.

Link between parasite and schizophrenia

The “crazy cat lady” has practically become a social stereotype in the United States and other countries, conjuring the image of a woman who lives with 25 cats and talks to herself a lot. But researchers investigating schizophrenia have actually identified a potential link between people who are exposed to Toxoplasma infection and the manifestations of schizophrenia; for example, several studies have identified higher levels of antibodies to the parasite in people with schizophrenia, and infection with Toxoplasma can cause damage to brain cells that is similar to the damage seen in patients with schizophrenia. Toxoplasmosis can also sometimes lead to symptoms of psychosis.

The fact is that most people don’t know they have toxoplasmosis because they have healthy immune systems. In people with compromised immunity, however, such as those with HIV, T. gondii can precipitate an extreme form of dementia that eventually kills them. The dementia is so severe that the sufferer eventually becomes completely unaware of his or her surroundings and lapses into a coma. The bug, however, also can affect the central nervous system in healthy people and is also linked to severe eye problems even in patients who are not immunocompromised. One researcher has claimed that infection with the parasite makes men dumber and women act like “sex kittens.”

ROP18: Watch out for this one

There are different strains of T. gondii, and investigators have noted that the Type 1 strain is most closely associated with disease. Studies of T. gondii, which has a genome with about 6000 genes, have pinpointed the virulence capacity of the strain to a single gene, dubbed ROP18. This gene encodes a kinase, one of a huge class of cell signaling proteins that add phosphates to molecules. Typically in cell signaling, kinases exist in a series, phosphorylating the next protein in the pathway, which helps maintain regulation of the signaling. The most virulent T. gondii strains have a form of the gene that differs from that carried by benign strains. Researchers speculate that this kinase interferes with a cell’s normal signaling, hijacking it for its own purposes, including growth and reproduction. The good news is that because kinases are so important in cell signaling, pharmaceutical companies have developed libraries of molecules that inhibit specific kinases, so one potential path to preventing toxoplasmosis is to discover an inhibitor of ROP18.

Rats get a little nutty from it, too

Not only has this parasite been linked to the ability to alter human behavior, but it also appears to alter rodent behavior in ways that favor its own reproduction. For example, rodents exposed to toxoplasma via cat feces actually become more likely to hang out near cat urine. If a cat eats the infected animal, the toxoplasmosis bug can then move into the sexual phase of its life cycle in the cat.

Rats are fast, cheap TB detectors

An unpredictable killer continues to kill

Can you name the disease that killed Chopin, Keats, Descartes, Kafka, Florence Nightingale, Eleanor Roosevelt and 200 million more people in the last 100 years? It’s tuberculosis, formerly known as “consumption,” and now known as TB. The tuberculosis bacterium, Mycobacterium tuberculosis, is an airborne pathogen that can be passed on from people with active cases of TB and usually settles in the lungs, where it can flourish and cause infection. It is possible to have what is known as “latent TB,” a situation in which you harbor the bacteria, but do not manifest the disease and are not contagious. Worldwide, World Health Organization predicts that the numbers of people who die from TB will climb to 8 million by 2015.

Experts agree that generally, when TB is caught early and treated, it is curable. But with millions of people suffering from it—including people with suppressed immune systems, such as those with HIV infections—detecting every case of TB is a tough job. Current methods require three saliva samples taken over a two-day period to be prepped on a slide, stained, and examined by a trained technician for the presence of TB bacteria. A good technician can analyze about 20 samples per day; for the 8 million people who may have TB in 2015, it would take 1200 technicians working 365 days to identify them all. One thing desperately needed in nations without the funding for technicians is a fast, accurate, low-tech way to analyze samples for the presence of TB.

Bacteria, explosives…whatever

Enter the rat. Bart J.C. Weetjens, who is not a rat, but a scientist working for Apopo, a Belgian company based in Tanzania, had one of those “chance favors the prepared mind” moments. He realized that the Dutch word for tuberculosis, tering, means something along the lines of “that’s starting to smell like tar.” Given that traditional Chinese medicine includes using smell to diagnose TB, Weetjens concluded that a trained animal might be able to detect TB, much in the way a bomb-sniffing dog detects explosives. It just so happened that Weetjens’ company had already trained a native African giant pouched rat, Cricetomys gambianus, to use its olfactory faculties to sniff out land mines. Weetjens decided simply to substitute TB bacteria for explosives.

The rats were uniquely qualified for the job. Unlike most nocturnal predators, these animals have very small eyes, indicating a strong reliance on olfactory and aural senses. During the day, they seem blind, sniffing the air rather than looking around. They can grow as large as a cat, are omnivorous, easily tamed and trained, and great breeders; a single female can produce 10 litters of up to four young each year. Weetjens took his idea to the World Bank, which agreed to fund a complete study of the rats’ ability to sniff out TB.

HeroRats

Weetjens already reported preliminary results that indicate the rats may be a viable way to ID TB. The rats identified 77 percent of infected saliva samples, and 92 percent of cultured bacteria samples, with a false-positive (indicating bacteria where there were none) rate of 2 percent. The current human-based process has an accuracy rate of about 95 percent, but Weetjens figures that with several rats analyzing samples, the rats’ accuracy will match the humans’. According to Weetjens, the rats can analyze 126 samples in 20 minutes, making them a very cheap, fast diagnostic test. The analysis of 8 million samples that would have taken 1200 humans 365 days would take only two rats working the same period of time. The latest data indicate similar success rates.

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